S295: TACROLIMUS MODULATES CD4+ T-CELL PROLIFERATION AND FUNCTION VIA MTHFD2 IN IMMUNE THROMBOCYTOPENIA
Authors: LI-Ping Yang, Hai-Xia Fu, Qiu-Sha Huang, Peng Zhao, Chen-Cong Wang, Yuan-Yuan Zhang, Xiao-Jun Huang, Xiao-Hui Zhang
Journal: HemaSphere (2023)

Topic: 32. Platelet disorders Background: Primary immune thrombocytopenia (ITP) is an autoimmune disease characterized by excessive platelet destruction and decreased platelet production. T cell immune abnormalities have been implicated in the pathogenesis of ITP. However, the mechanisms are incompletely understood. Recent studies suggest that the one-carbon (1C) metabolism enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) in this pathway, controls effector and regulatory T cell fate and function. Targeting MTHFD2 reduces disease severity in several autoimmune diseases. Our previous data indicated that the dysregulated polarization of T helper cells was involved in the pathogenesis of ITP (J Thromb Haemost. 2023). Tacrolimus, which was demonstrated to be a promising therapeutic option for ITP patients in our clinical study (63th ASH, 2021), may modulate T cell proliferation and function via MTHFD2. Aims: This study was aimed at revealing the metabolic characteristics of T cells, exploring the role of MTHFD2 in T cell proliferation, activation and function, and investigating the underlying effects and molecular mechanisms of tacrolimus treatment in ITP patients. Methods: Peripheral blood samples were collected from 35 patients with primary ITP and 25 healthy donors (HDs). Time-of-flight mass cytometry was performed to quantify immune subsets and metabolic regulators associated with ITP at single-cell resolution. Seahorse XFe96 Extracellular Flux Analyzer and mass spectrometer were used for metabolic experiments. Additionally, an active ITP mouse model was established to observe the therapeutic effects of tacrolimus. Results: One-carbon metabolism, especially MTHFD2, was upregulated in CD4+ T cells from ITP patients relative to those from HDs. Additionally, the proliferation, as measured by CFSE, and activation, as measured by CD25 and CD69 expression, of CD4+ T cells were reduced after MTHFD2 inhibitor (MTHFD2i) treatment. Flow cytometry and RT-PCR showed that MTHFD2i reduced the expression of T-bet and RORγt while significantly increasing the percentage of CD4+CD25+FoxP3+ Treg cells in ITP. The proinflammatory cytokine IL-17 was also suppressed by MTHFD2i. The extracellular acidification rate (ECAR), an indicator of aerobic glycolysis, was increased, while the oxygen rate (OCR), a proxy for mitochondrial oxidative phosphorylation (OXPHOS), was reduced in CD4+ T cells from ITP patients compared with those from HDs. MTHFD2i increased the OCR/ECAR ratio, promoting a shift towards OXPHOS. Moreover, MTHFD2i inhibited purine biosynthesis and depleted intermediates of the TCA cycle in CD4+ T cells from patients with ITP. Similar findings were observed in CD4+ T cells from mice with ITP. To reveal the therapeutic effect of tacrolimus and the underlying mechanism, we found that both patients and mice with ITP had elevated platelet counts and alleviated bleeding after treatment with tacrolimus. Similar to MTHFD2i, tacrolimus inhibited the proliferation and proinflammatory cytokine production of CD4+ T cells. Additionally, tacrolimus suppressed the expression of MTHFD2 in CD4+ T cells from ITP mice. Taken together, these results showed that tacrolimus modulated the proliferation and function of T cells via MTHFD2. Furthermore, tacrolimus induced metabolic reprogramming by inhibiting glycolysis and increasing OXPHOS in CD4+ T cells. Summary/Conclusion: Our data unveil how dysregulated 1C metabolism controls T cell survival, activation and effector function in ITP and suggest a novel therapeutic opportunity of tacrolimus towards targeting MTHFD2. Keywords: T cell, Tacrolimus, Immune thrombocytopenia (ITP)